Peptidergic neurons are found in great numbers in the gut. Evidence from our recent experiments and those of others indicates that the environment in general and the gut in particular can influence the expression of peptide phenotypes. We propose to investigate the elements in the gut environment that are critical for influencing peptide expression. Using in vivo and in vitro culture techniques, we will study the production of neuropeptides in quail ciliary ganglion-chick aneural gut combinations. Ciliary ganglion neurons in situ contain only acetylcholine and the neuropeptide somatostatin. We will study with in vivo culture methods the ability of aneural gut to promote expression of neuropeptides in ciliary ganglion-gut tissue combinations grown on the chorioallantoic membrane of host chick embryos. This approach will permit an evaluation of the variables of gut age, gut region, and gut innervation on the production of neuropeptides in a setting where the three-dimensional tissue integrity and the hormonal milieu of the embryo are preserved. Using in vitro methods we will analyze the expression of neuropeptides in ciliary ganglia grown in culture media with gut of different ages. This approach will permit us to determine if a soluble factor or cell-to-cell contact is important to peptide expression. In a last set of experiments the ability of the gut and tissue extracts to influence peptide expression in mesencephalic neural crest cultures will be studied. A portion of the mesencephalic neural crest is a precursor for the ciliary ganglion. Peptides including vasoactive intestinal peptide, somatostatin, enkephalin and substance P will be localized by immunocytochemistry and measured by radioimmunoassay. Because there may be a link between neuropeptide and cholinergic expression, we will study the possible co-localization of neuropeptides and ChAT, an enzyme marker specific for cholinergic neurons. With this combination of techniques, we will elucidate the elements in the gut environment that influence neuropeptide expression. The information obtained here will be useful in understanding the underlying defects that lead to clinical conditions such as Hirschsprung's disease and pseudo-obstruction.